Prodrugs for use as ophthalmic agents

ABSTRACT

The subject invention provides a mechanism by which steroidal quinol compounds confer beneficial ophthalmic effects. The subject compounds possess a lipophilic-hydrophilic balance for transcorneal penetration and are readily reduced into parent phenolic A-ring steroid compounds to provide protection or treatment against various ocular symptoms and disorders. The compounds according to the subject invention appear to be highly advantageous as prodrugs to provide protection and/or treatment against ocular disorders. These prodrugs confer lipid solubility optimal for transocorneal penetration and are readily converted to endogenous reducing agents into active phenolic A-ring steroid compounds. To the extent that these prodrugs have reduced feminizing effects and systemic toxicity, they would be expected to be quite advantageous for protecting or treating the eye against ocular disorders such as cataract or glaucoma without undesired (systemic) side effects).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. Ser. No.10/405,413, filed Apr. 1, 2003, now U.S. Pat. No. 7,026,306; whichclaims the benefit of U.S. provisional patent application Ser. No.60/369,589, filed Apr. 1, 2002. This Application also claims the benefitof U.S. provisional patent application Ser. No. 60/432,354, filed Dec.9, 2002.

FIELD OF THE INVENTION

The present invention relates to prodrugs for use as ophthalmic agents,specifically for retinal protection. In particular, the presentinvention relates to the use of steroidal quinols as prodrugs ofphenolic A-ring steroid compounds to treat and/or prevent eyepathologies.

BACKGROUND OF INVENTION

A variety of tissues metabolize estrogen (as a representative phenolicA-ring steroid) to various degrees. Of all of the tissues investigated,cornea appears to be the most active estrogen-metabolizing tissue(Stàrka, L and J Obenberger, “In vitro Estrone-Estradiol-17βInterconversion in the Cornea, Lens, Iris and Retina of the Rabbit Eye,”Arch Klin Exp Ophthalmol, 196:199–204 (1975)). Estrogens havedemonstrated an important role in the health maintenance of all mucousmembranes in the body, including the maintenance of a healthy ocularsurface. Additional studies have revealed that the biological activityof estrogen may be effective in the protection and treatment of the eye,including the lens and retina, against cataracts and the detrimentaleffects of glaucoma.

Unfortunately, many regions of the eye are relatively inaccessible tosystemically administered estrogens. For example, orally administeredestrogen passes through the liver before reaching estrogen sensitivetissues. Because the liver contains enzymes that can inactivate theestrogen, the estrogen that eventually reaches tissue targeted fortreatment is virtually ineffective. Moreover, systemic administration ofestrogen often produces undesirable side effects, i.e., feminizing sideeffects in men.

As a result, topical drug delivery remains the preferred route ofadministration to the eye. There are a variety of factors that affectthe absorption of drugs into the eye. These factors include: theinstillation volume of the drug, the frequency of instilled drugadministration, the structure and integrity of the cornea, the proteinlevel in tears, the level of enzymes in tears, lacrimal drainage andtear turnover rate, as well the rate of adsorption and absorption of adrug by the conjunctiva, sclera, and eyelids.

Thus, the potential treatment of ocular disorders/conditions byestrogens or agents derived from estrogens is confounded by poor ocularbioavailability of pharmacologically active agents and by the likelihoodof triggering systemic side effects associated with the administrationof natural (endogenous) estrogens. The latter are due to absorption fromthe nasal cavity and the gastrointestinal (GI) tract after the topicallyadministered estrogen hormone gains access to these pathways through itsremoval by the nasolacrimal apparatus of the eye. A potential way ofreducing or even eliminating systemic side effects is to improve oculartargeting that would allow for the use of reduced doses of thebiologically active agent in the ophthalmic drug formation.

Accordingly, the direct administration to an eye lens of estrogen havingquinolines (i.e., 6-hydroxyquinoline) and fused quinolines that act assteroid receptor modulators to prevent or treat cataract disorders hasbeen disclosed. In addition, the administration of 17-β-estradiol to thesurface of the eye to alleviate dry-eye syndrome or keratoconjunctivitissicca has been disclosed. Glycosides of catechol estrogens have beenformulated that demonstrate antioxidant activity to the same degree asto that of the parent catechol estrogens. Nonetheless, all of thepreviously disclosed compounds and methods for applying estrogens to theeye relate to compounds that lack efficient corneal penetration and/orare unapplicable to men because of their activity as a female hormone.

As noted above, the major barrier to ocular drug penetration is thecornea. The cornea is composed of three layers: a lipid-rich epithelium,a lipid-poor soma, and a lipid-rich endothelium. Therefore, an agentmust possess both lipophilic-hydrophilic balance for adequatetranscorneal penetration and, thus, ocular bioavailability (Akers H J,“Ocular bioavailability of topically applied ophthalmic drugs,” AmPharm, NS23:33–36 (1983)). Thus, poor ocular bioavailability is an issuefor estrogens and their synthetic analogs, because estrogens are highlylipid soluble molecules that are usually not amenable to adequatetranscorneal penetration.

Prodrugs are inactive compounds that are converted in vivo intobiologically active agents by enzymatic and/or chemical transformations.Prodrugs are advantageous because they can be designed to overcomeproblems associated with stability, toxicity, lack of specificity, orlimited bioavailability, that may exist with the active form of a drug.Thus, there is a need to develop effective prodrugs of estrogen as amedical compound.

Estrogen quinols have been known for decades among organic chemists(Gold A. M., and Schwenk E., “Synthesis and reaction of steroidalquinols,” J Am Chem Soc, 80:5683–5687 (1958)) though their metabolicformation has only been reported recently (Ohe T., et al., “Novelmetabolic pathway of estrone and 17β-estradiol catalyzed by cytochromeP-450”, Drug Metab Dispos, 28:11–112 (2000)).10β-hydroxy-1,4-estradiene-3,7-dione and10β,17β-dihydroxy-1,4-estradiene-3-one were detected from the respectiveestrogens during metabolic oxidation catalyzed by several cytochromeP-450 isoenzymes in rat liver microsomal systems. Contrary to well-knowncatechol metabolites of estrogens (Zhu, B. T. and Conney A. H.,“Functional role of estrogen metabolism in target cells: review andperspective,” Carcinogenesis, 19:1–27 (1998)), quinols do not possess anaromatic A-ring, making their biochemistry substantially different fromthat of catechols. Studies are currently underway to assess the natureof estrogen quinols.

BRIEF SUMMARY

The subject invention provides materials and methods wherein unique andadvantageous steroidal quinols are used for a broad range of therapeuticpurposes, including the treatment or prevention of ophthalmic disordersand/or conditions by modulating or activating estrogen receptors. Thesedisorders and/or conditions include, but are not limited to,conjunctivitis, diabetic retinopathy, dry eye, glaucoma, and cataract.

A quinol (i.e., the 10α,β-hydroxyestra-1,4-diene-3-one structures)derived synthetically from phenolic A-ring steroids has been found toconfer significant reduced lipid solubility compared to the parentphenolic A-ring steroid compounds to provide improved transcornealpenetration. Further, these quinols can be converted to phenolic A-ringsteroid structures by endogeneous NAD(P)H as a reducing agent. In oneembodiment, an oxidoreductase catalyst converts subject steroidalquinols to phenolic A-ring steroids that possess pharmacologicalactivity in the eye. The present invention exploits the benefits ofprodrugs (including but not exclusively based on the quinol structure asa novel pro-moiety) for phenolic A-ring steroid compounds to provideocular bioavailability of the therapeutic agent in question. Prodrugsare, by definition, inactive compounds that are converted to thebiologically active agents by chemical or enzymatic transformation invivo.

The subject invention provides a mechanism by which quinol derivedphenolic A-ring steroid compounds confer beneficial ophthalmic effects.The subject compounds possess a lipophilic-hydrophilic balance fortranscorneal penetration and are readily reduced into parent phenolicA-ring steroid compounds to provide protection or treatment againstvarious ocular symptoms and disorders. The compounds according to thesubject invention appear to be highly advantageous as prodrugs toprovide protection and/or treatment against ocular disorders. Theseprodrugs confer low lipid solubility and are readily converted byendogenous reducing agents into active phenolic A-ring steroidcompounds. To the extent that these prodrugs have reduced feminizingeffects and systemic toxicity, they would be expected to be quiteadvantageous for protecting or treating the eye against ocular disorderssuch as cataract or glaucoma.

In a specific embodiment, the subject invention provides steroidalquinol compounds that are, themselves, inactive. However, these quinolstructures can act as prodrugs because they are converted to atherapeutically active phenolic A-ring steroid upon exposure to areducing agent. Additionally, because an active phenolic A-ring steroidcompound arises after conversion by a reducing agent, a smallerconcentration of the steroidal quinols is required as compared to directadministration of phenolic A-ring steroid, thus reducing the potentialfor systemic toxicity.

In one particular embodiment of the subject invention, isomers of10-hydroxyestra-1,4-diene-3-one quinol structure (estrone-quinol) areconverted to active, phenolic A-ring steroid compounds (i.e., estrone)when exposed to a reducing agent. In related embodiments, quinols arederived from estrogen analogues, i.e.,3,17-dihydroxyestra-1,3,5(10),9(11)-tetraene (ZYC1).

In another embodiment, steroidal quinols are provided as prodrugs thatrequire at least one-step activation in vivo to yield pharmaceuticallyactive estrogen compounds. In a related embodiment, quinols derived fromestrogen prodrugs that require two-step activation can include a polarfunctional group to enhance hydrophilicity at the 17-OH group or mayhave the 10-OH group esterified to decrease lipophilicity throughphosphate, or N,N,N-trialkylammonium esters.

In another embodiment, the 3,17-keto groups of quinols of the presentinvention can be functionalized as oxime and/or alkoximes. In doing so,preliminary compounds to the subject quinols are created (to form i.e.pro-prodrugs). Such functionalized quinols (i.e., 3-keto functionalizedas an oxime) can be used for a variety of therapeutic purposes,including use for ocular-specific delivery of phenolic A-ring steroids.

An object of the present invention is to provide compounds formulatedfor ophthalmic administration. For example, solutions or suspensions ofthese compounds may be formulated in the form of eye drops, ormembranous ocular patch, which is applied directly to the surface of theeye.

It is another object of the subject invention to provide an ophthalmicagent with an increased therapeutic index associated with treatmentsusing the subject compounds disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the viability of retinal ganglial cells in thepresence of glutamate, estrogen analog3,17-dihydroxyestra-1,3,5(10),9(11)-tetraene (ZYC1), or combinations ofglutamate and various concentrations of ZYC1.

FIG. 2 illustrates retinal ganglial cell viability when treated withglutamate in the presence or absence of ZYC1 or ZYC1 incubated in thepresence of various concentrations of estrogen receptor antagonist,ICII82,780 (ICI).

FIG. 3 illustrates a quinol acetate in accordance with the subjectinvention.

FIG. 4 illustrates an (alk)oxime of a quinol, in accordance with thesubject invention.

FIG. 5 illustrates the viability of retinal ganglial cells in thepresence of glutamate, phenolic A-ring steroid2-(1-adamantyl)-3-hydroxyestra-1,3,5(10)-trien-17-one (ZYC3), orcombinations of glutamate and various concentrations of ZYC3.

FIG. 6 illustrates LC/MS/MS analysis demonstrating the detection of10β-hydroxyestra-1,4-dien-3,17-dione (estrone-quinol, t_(R)=1.38) and aproduct formed from it (t_(R)=4.5 min.) after the incubation ofestrone-quinol with NADPH.

FIG. 7 illustrates MS/MS analysis of the chromatographic peak att_(R)=4.5 min., which is identical to that of estrone.

FIG. 8 illustrates MS³ recording from the chromatographic peak,t_(R)=4.5 min., m/z 253 selected as precursor after MS/MS, which isidentical to that of estrone.

DETAILED DISCLOSURE

The subject invention provides steroidal quinol compounds that producephenolic A-ring steroids in vivo. In one embodiment, these compoundsprovide improved physicochemical properties including, but not limitedto, favorable ocular bioavailability and facile transcornealpenetration. In a preferred embodiment, estrogen derived quinolcompounds demonstrate decreased lipophilicity as compared to lipophilicestrogens and estrogen analogues.

In another embodiment of this invention, these compounds treat and/orprotect against various ocular diseases. Preferred compounds of thesubject invention are effective in treating and/or preventing maladiesassociated with vision-threatening intraocular damage due topathophysiological predispositions. Particularly preferred compounds arethose which treat glaucoma and/or macular degeneration.

In a specific embodiment, the subject invention provides steroidalquinol compounds that are, themselves, inactive. However, these quinolstructures can act as prodrugs because they are converted to atherapeutically active phenolic A-ring steroid upon exposure to areducing agent. Additionally, because an active phenolic A-ring steroidcompound arises after conversion by a reducing agent, a smallerconcentration of the steroidal quinols is required due to their improvedocular bioavailability as compared to direct administration of estrogen,thus reducing the potential for systemic toxicity. In a relatedembodiment of the subject invention, steroidal quinols are provided asprodrugs that are converted into an active phenolic A-ring steroid via aone-step conversion by a reducing agent. Suitable reducing agentsinclude endogenous NAD(P)H or oxidoreductases.

In one particular embodiment of the subject invention, a10β-hydroxyestra-1,4-diene-3-one quinol structure (estrone-quinol) isconverted to an active, phenolic A-ring estrogen compound (estrone) whenexposed to a reducing agent. In related embodiments, quinols are derivedfrom estrogen analogues, i.e.,3,17-dihydroxyestra-1,3,5(10),9(11)-tetraene (ZYC1) or2-(1-Adamantyl)estrone (ZYC3).

In another embodiment, steroidal quinols are provided as prodrugs thatrequire two (or more than two) step activation in vivo to yieldpharmaceutically active estrogen compounds. The liberation of a parentestrogen occurs through a two-step reaction: (1) enzymatic (phosphatase,esterase) cleavage of the ester group followed by (2) spontaneous andfast chemical conversion of a quinol by an endogenous reducing agent. Ina related embodiment, these compounds according to the present inventioncan include a polar functional group to enhance hydrophilicity at the17-OH group or may have the 10-OH group esterified to decreaselipophilicity through phosphate or N,N,N-trialkylammonium esters.

In another embodiment, the prodrugs according to the subject inventioncan be synthesized by attaching a polar functional group to enhanceaffinity to water and facilitate the transport of the prodrug of thesubject invention through the lipid-poor middle stroma in the cornea. Ina preferred embodiment, the 17-OH group of a quinol according to thesubject invention is the primary site to which a polar functional groupis added. In another preferred embodiment, the 10β-OH of a steroidalquinol (i.e., 17-hydroxyestra-1,4-diene-17-one) is blocked byesterification to make the resultant prodrug more lipophilic than thephenolic A-ring steroid derived quinol.

It will be noted that the structure of some of the compounds of thisinvention includes asymmetric carbon atoms. It is to be understoodaccordingly that the isomers arising from such asymmetry (i.e., allenantiomers and diastereomers) are included within the scope of thisinvention, unless indicated otherwise. Such isomers can be obtained insubstantially pure form by conventional methods including, for example,by classical separation techniques and by stereochemically controlledsynthesis.

DEFINITIONS

A number of terms are used herein to designate particular elements ofthe present invention. When so used, the following meanings areintended:

The term “estrogen,” as used herein, refers to both naturally occurringand synthetic substances classed as estrogen on the basis of theirtherapeutic or biological action (see listing under ‘Estrogens’ in the‘Therapeutic Category and Biological Activity Index’ of The Merck Index,12th Edition, Merck Research Laboratories, NJ, 1996, page THER-22).According to this listing, estrogens may be steroids (i.e., estradiol,ethinyl estradiol, colpormon, conjugated estrogenic hormones, equilenin,equilin, estriol, estrone, mestranol, moxestrol, mytatrienediol,quinestradiol and quinestrol) or non-steroids (i.e., diethylstilbestrol,dienestrol, benzestrol, broparoestrol, chlorotrianisene, dimestrol,fosfestrol, hexestrol, methallenestril, methestrol). Additionalsubstances known to be estrogenic, that is, they interact with cellularestrogen receptors and mimic the effects of estrogens, includeestrogenic substances that have been shown to be tissue selective intheir estrogenic effects. Diverse classes of molecules fall within thiscategory, for example: quinolines and fused quinolines that act assteroid receptor modulators such as3,9-dihydroxy-5H-benzofuro[3,2-c]quinoline-6-one and those disclosed inWO 96/19458; phytoestrogens which occur naturally in plants such asforage plants, soya beans, seeds, berries and nuts (Jordan et al.,“Structure-activity relationships of estrogen,” Env. Health Per.,61:97–110 (1985)), including isoflavones such as genistein and genisteinglycosides, equol, O-desmethyl-angolensin, biochanin A, daidzein andformononetin; flavones such as phloretin, 4′-6-dihydroxyflavone andtricin, and coumestans such as coumestrol, 4′-O-methyl coumestrol,medicagol and sativol, lignans such as matairesinol, enterodiol,enterolactone, guaiaretic acid, nordihydroguaiaretic acid andderivatives thereof, β-sitosterol; mycoestrogens such as zeranol,zearalenol and zearalenone; estrogen receptor agonist/antagonists, suchas tamoxifen, hydroxytamoxifen, zindoxifene and its metabolites,nafoxidene and derivatives, clomiphene, centchroman, benzothiophenes andrelated compounds such as benzothiophene-derived LY139478 (Eli Lilly),raloxifene and droloxifene, which may mimic the action of estrogens incertain types of cells, while opposing it in others (Raisz, L. G.,“Estrogen and bone: new pieces to the puzzle,” Nature. Med.,2(10):1077–8 (1996)); and many para-substituted phenols that contain astrategically located phenolic hydroxyl not impaired by an alkylsubstitution (see Jordan et al., “Structure-activity relationships ofestrogen,” Env. Health Per., 61:97–110 (1985)), including octyl phenyl,nonyl phenol, butylated hydroxyanisole, bisphenol A andtrihydroxy-8-prenylflavone. Note that estrogenic substances in thisgeneral category may also be referred to in the literature as‘estrogens’ (see Jordan et al., 1985, for example). As already describedabove (for ‘estrogens’ as defined in Merck), estrogenic substances mayexert their estrogenic effect(s) directly or they may require metabolicconversion to an active form after administration. For example,metabolic activation of some phytoestrogens involves demethylation tophenols (Jordan et al., “Structure-activity relationships of estrogen,”Env. Health Per., 61:97–110 (1985)).

The term “estrogen derived quinols,” (i.e.,10α/β-hydroxyestra-1,4-diene-3-one structure) as used herein, refers toquinols and quinol derivatives related to estrogens, as described above,and para-substituted phenols obtained by oxidation of the phenolic ring,as described below.

The term “phenolic A-ring steroid” used herein refers to compoundscontaining a 3-hydroxy-1,3,5(10)-triene moiety as the six-memberedA-ring of a steroid, steroid analogue or steroid mimic, includingcompounds that manifest affinity to estrogen receptors (i.e.,3,17-dihydroxyestra-1,3,5(10),9(11)-tetraene) as well as compounds thatdo not bind to such receptors (i.e.,2-(1-adamantyl)-3-hydroxyestra-1,3,5(10)-trien-17-one).

The term “steroidal quinol” used herein refers to a steroid containing a10α/β-hydroxy-1,4-diene-3-one moiety as the six-membered A-ring of asteroid, steroid analogue or steroid mimic.

The term “ophthalmic disorders,” and/or “ophthalmic conditions,” as usedherein, refers to ophthalmic diseases, conditions, and/or disordersincluding, without limitation, those associated with the anteriorchamber of the eye (i.e., hyphema, synechia); the choroid (i.e.,choroidal detachment, choroidal melanoma, multifocal choroidopathysyndromes); the conjunctiva (i.e., conjunctivitis, cicatricialpemphigoid, filtering Bleb complications, conjunctival melanoma,Pharyngoconjunctival Fever, pterygium, conjunctival squamous cellcarcinoma); connective tissue disorders (i.e., ankylosing spondylitis,pseudoxanthoma elasticum, corneal abrasion or edema, limbal dermoid,crystalline dystrophy keratits, keratoconjunctivitis, keratoconus,keratopathy, megalocornea, corneal ulcer); dermatologic disorders (i.e.,ecrodermatitis enteropathica, atopic dermatitis, ocular rosacea,psoriasis, Stevens-Johnson syndrome); endocrine disorders (i.e.,pituitary apoplexy); extraocular disorders (i.e., Abducens Nerve Palsy,Brown syndrome, Duane syndrome, esotropia, exotropia, oculomotor nervepalsy); genetic disorders (i.e., albinism, Down syndrome, PetersAnomaly); the globe (i.e., anophthalmos, endophthalmitis); hematologicand cardiovascular disorders (i.e., Giant Cell Arteritis, hypertension,leukemias, Ocular Ischemic syndrome, sickle cell disease); infectiousdiseases (i.e., actinomycosis, botulism, HIV, diphtheria, Escherichiacoli, Tuberculosis, ocular manifestations of syphilis); intraocularpressure (i.e., glaucoma, ocular hypotony, Posner-Schlossman syndrome),the iris and ciliary body (i.e., aniridia, iris prolaps, juvenilexanthogranuloma, ciliary body melanoma, iris melanoma, uveitis); thelacrimal system (i.e., alacrima, Dry Eye syndrome, lacrimal glandtumors); the lens (i.e., cataract, ectopia lentis, intraocular lensdecentration or dislocation); the lid (i.e., blepharitis,dermatochalasis, distichiasis, ectropion, eyelid coloboma, Floppy Eyesyndrome, trichiasis, xanthelasma); metabolic disorders (i.e., gout,hyperlipoproteinemia, Oculocerebrorenal syndrome); neurologic disorders(i.e., Bell Palsy, diplopia, multiple sclerosis); general ophthalmologic(i.e., red eye, cataracts, macular degeneration, red eye, maculardegeneration); the optic nerve (i.e., miningioma, optic neuritis, opticneuropathy, papilledema); the orbit (i.e., orbital cellulits, orbitaldermoid, orbital tumors); phakomatoses (i.e., ataxia-telangiectasia,neurofibromatosis-1); presbyopia; the pupil (i.e., anisocoria, Hornersyndrome); refractive disorders (i.e., astigmatism, hyperopia, myopia);the retina (i.e., Coats disease, Eales disease, macular edema,retinitis, retinopathy); and the sclera (i.e., episcleritis, scleritis).

The term “patient,” as used herein, describes an organism, includingmammals, to which treatment with the compositions according to thepresent invention is provided. Mammalian species that benefit from thedisclosed methods of treatment include, and are not limited to, apes,chimpanzees, orangutans, humans, monkeys; and domesticated animals(i.e., pets) such as dogs, cats, mice, rats, guinea pigs, and hamsters.

The term “polar aprotic solvent” refers to polar organic solventslacking an easily removed proton, including, but not limited to, ethylacetate, dimethylformamide (DMF), and acetonitrile.

The term “pharmaceutically acceptable salts,” as used herein, refers tothose carboxylate salts, esters, and prodrugs of the compound of thepresent invention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the invention.

Pharmaceutically acceptable salts are well known in the art and refer tothe relatively non-toxic, inorganic and organic acid addition salts ofthe compound of the present invention. For example, S. M. Berge, et al.describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66:1–19 (1977) which is incorporated herein byreference. The salts can be prepared in situ during the final isolationand purification of the compounds of the invention, or separately byreacting the free base function with a suitable organic acid. Examplesof pharmaceutically acceptable, nontoxic acid addition salts are saltsof an amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate,laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

The term “pharmaceutically acceptable prodrugs,” as used herein, refersto those prodrugs of the compounds of the present invention which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response, and the like, commensurate with areasonable benefit/risk ratio, and effective for their intended use, aswell as the zwitterionic forms, where possible, of the compounds of theinvention.

The term “prodrug,” as used herein, refers to a derivative of abiologically active compound (i.e., the steroidal quinols according tothe present invention) that lacks pharmaceutical activity, but isconverted (i.e., by NAD(P)H) to an active agent, which is a phenolicA-ring steroid such as estrogen hormone, estrogen analogue, substitutedestrogen or estrogen-receptor agonist or antagonist) upon interactionwith a biological or chemical system, for example catalyzed reduction byenzymes in the eye. A thorough discussion is provided in T. Higuchi andV. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S.Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers inDrug Design, American Pharmaceutical Association and Pergamon Press,1987, both of which are incorporated herein by reference. A prodrug,according to the present invention, can be converted into an activecompound with one or more steps.

The term “substituted” shall be deemed to include multiple degrees ofsubstitution by a named substituent. Where multiple substituent moietiesare disclosed, the substituted compound can be independently substitutedby one or more of the disclosed or claimed substituent moieties, singlyor severally.

Unless otherwise specified, as used herein, the term “alkyl” refers to astraight or branched or cyclic alkyl moiety. In one embodiment, thealkyl moiety is C₁₋₂₀ alkyl, which refers to an alkyl moiety having fromone to twenty carbon atoms, including for example, methyl, ethyl,propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl and octyl,cycloalkyl including for example cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl. The alkyl group specifically includes fluorinated alkylssuch as CF₃ and other halogenated alkyls such as CH₂CF₂, CF₂CF₃, thechloro analogs, and the like. The alkyl group can be optionallysubstituted with one or more moieties selected from the group consistingof aryl, heteroaryl, heterocyclic, carbocycle, alkoxy, heterocycloxy,heterocylalkoxy, aryloxy; arylalkoxy; heteroaryloxy; heteroarylalkoxy,carbohydrate, amino acid, amino acid esters, amino acid amides, alditol,halo, haloalkyl, hydroxyl, carboxyl, acyl, acyloxy, amino, amido,alkylamino, dialkylamino, arylamino, nitro, cyano, thiol, imide,sulfonic acid, sulfate, sulfonyl, sulfanyl, sulfinyl, sulfamoyl,carboxylic ester, carboxylic acid, amide, phosphonyl, phosphinyl,phosphoryl, thioester, thioether, oxime, hydrazine, carbamate,phosphonic acid, phosphate, phosphonate, phosphinate, sulfonamido,carboxamido, hydroxamic acid, sulfonylimide, substituted orunsubstituted urea connected through nitrogen; or any other desiredfunctional group that does not inhibit the pharmacological activity ofthis compound, either unprotected, or protected as necessary, as knownto those skilled in the art, for example, as taught in Greene, et al.,Protective Groups in Organic Synthesis, John Wiley and Sons, SecondEdition, 1991, hereby incorporated by reference.

The term “alkenyl” refers to a straight or branched alkyl moiety havingone or more carbon double bonds, of either E or Z stereochemistry whereapplicable. This term includes for example, vinyl, 1-propenyl, 1- and2-butenyl, and 2-methyl-2-propenyl, as well as “cycloalkenyl” groupssuch as cyclopentenyl and cyclohexenyl.

The term “alkoxy,” as used herein, and unless otherwise specified,refers to a moiety of the structure —O-alkyl, wherein alkyl is asdefined above. The alkyl group can be optionally substituted asdescribed above. Alkoxy groups can include OCF₃, OCH₂CF₃, OCF₂CF₃, andthe like.

The term alkynyl refers to a hydrocarbon with at least one triple bond,including for example, C₁ to C₁₀ groups including but not limited toethynyl, 1-propynyl, 1- and 2-butynyl, 1-methyl-2-butynyl, and the like.

The term “aryl,” as used herein, and unless otherwise specified, refersto phenyl, biphenyl, or naphthyl, and preferably phenyl. The aryl groupcan be optionally substituted with one or more of the moieties selectedfrom the group consisting of alkyl, heteroaryl, heterocyclic,carbocycle, alkoxy, aryloxy, aryloxy; arylalkoxy; heteroaryloxy;heteroarylalkoxy, carbohydrate, amino acid, amino acid esters, aminoacid amides, alditol, halo, haloalkyl, hydroxyl, carboxyl, acyl,acyloxy, amino, amido, alkylamino, dialkylamino, arylamino, nitro,cyano, thiol, imide, sulfonic acid, sulfate, sulfonyl, sulfanyl,sulfinyl, sulfamoyl, carboxylic ester, carboxylic acid, amide,phosphonyl, phosphinyl, phosphoryl, thioester, thioether, oxime,hydrazine, carbamate, phosphonic acid, phosphate, phosphonate,phosphinate, sulfonamido, carboxamido, hydroxamic acid, sulfonylimide orany other desired functional group that does not inhibit thepharmacological activity of this compound, either unprotected, orprotected as necessary, as known to those skilled in the art, forexample, as taught in Greene, et al., “Protective Groups in OrganicSynthesis,” John Wiley and Sons, Second Edition, 1991. Alternatively,adjacent groups on the aryl ring may combine to form a 5 to 7 memberedcarbocyclic, aryl, heteroaryl or heterocylic ring.

The term “aralkoxy” refers to an aryl group attached to an alkyl groupthat is attached to the molecule through an oxygen atom. The aryl andalkyl groups can be optionally substituted as described above.

The term “aralkyl,” as used herein, and unless otherwise specified,refers to an aryl group as defined above linked to the molecule throughan alkyl group as defined above. The aryl and alkyl portions can beoptionally substituted as described above.

The term “aryloxy,” as used herein, refers to an aryl group bound to themolecule through an oxygen atom. The aryl group can be optionallysubstituted as set out above for aryl groups. The terms “heteroaryl” and“heteroaromatic,” as used herein, refer to monocyclic or bicyclicaromatic ring systems of five to ten atoms of which at least one atom isselected from O, N, and S, in which a carbon or nitrogen atom is thepoint of attachment, and in which one additional carbon atom isoptionally replaced with a heteroatom selected from O or S, and in whichfrom 1 to 3 additional carbon atoms are replaced by nitrogenheteroatoms.

Heteroaryl thus includes aromatic and partially aromatic groups thatcontain one or more heteroatoms. Examples of this type include but arenot limited to are furan, benzofuran, thiophene, benzothiophene,pyrrole, pyrazole, imidazole, oxazole, benzoxazole, thiazole,benzthiazole, isothiazole, thiadiazole, triazole, benzotriazole,furazan, benzofurazan, thiafurazan, benzothiafurazan, tetrazole,oxadiazole, triazine, pyridine, pyridazine, pyrimidine, pyrazine,triazine, indolizine, indole, isoindole, purine, quinoline,benzimidazole, pteridine, isoquinoline, cinnoline, quinazoline, andquinoxaline.

The term “heteroaralkyl,” as used herein, and unless otherwisespecified, refers to a heteroaryl group as defined above linked to themolecule through an alkyl group as defined above.

The term “heterocyclealkyl,” as used herein, refers to a heterocyclicgroup bound to the molecule through an alkyl group. The heterocyclicgroup and the alkyl group can be optionally substituted as describedabove. The term “heterocycloalkyl” can also refer to a saturatedheterocyclic moiety having from two to six carbon atoms and one or moreheteroatom from the group N, O, and S (or oxidized versions thereof)which may be optionally benzofused at any available position. Thisincludes, for example, azetidinyl, pyrrolidinyl, tetrahydrofuranyl,piperidinyl, benzodioxolyl and the like. The term “heterocycloalkyl”also refers to an alicyclic moiety having from three to six carbon atomsand one or more heteroatoms from the group N, O, and S and having inaddition one double bond. Such moieties may also be referred to as“heterocycloalkenyl” and includes, for example, dihydropyranyl, and thelike.

The term “heterocyclic” refers to a nonaromatic cyclic group that may bepartially (contains at least one double bond) or fully saturated andwherein there is at least one heteroatom, such as oxygen, sulfur,nitrogen, or phosphorus in the ring. The term heteroaryl orheteroaromatic, as used herein, refers to an aromatic that includes atleast one sulfur, oxygen, nitrogen or phosphorus in the aromatic ring.Nonlimiting examples of heterocylics and heteroaromatics arepyrrolidinyl, tetrahydrofuryl, piperazinyl, piperidinyl, morpholino,thiomorpholino, tetrahydropyranyl, imidazolyl, pyrolinyl, pyrazolinyl,indolinyl, dioxolanyl, 1,4-dioxanyl, aziridinyl, furyl, furanyl,pyridyl, pyrimidinyl, benzoxazolyl, 1,2,4-oxadiazolyl,1,3,4-oxadiazolyl, 1,3,4-thiadiazole, indazolyl, 1,3,5-triazinyl,thienyl, isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl, benzofuranyl,quinolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, indolyl,isoindolyl, benzimidazolyl, purinyl, carbazolyl, oxazolyl, thiazolyl,benzothiazolyl, isothiazolyl, 1,2,4-thiadiazolyl, isooxazolyl, pyrrolyl,quinazolinyl, cinnolinyl, phthalazinyl, xanthinyl, hypoxanthinyl,pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole,thiazine, pyridazine, or pteridinyl wherein a heteroaryl or heterocyclicgroup can be optionally substituted with one or more substituentselected from the same substituents as set out above for aryl groups.Functional oxygen and nitrogen groups on the heteroaryl group can beprotected as necessary or desired. Suitable protecting groups caninclude trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, andt-butyldiphenylsilyl, trityl or substituted trityl, alkyl groups, acylgroups such as acetyl and propionyl, methanesulfonyl, andp-toluenelsulfonyl.

The term “heteroaryloxy,” as used herein, refers to a heteroaryl groupbound to the molecule through an oxygen atom. The heteroaryl group canbe optionally substituted as set out above for aryl groups.

The term “heterocyclearalkoxy” refers to a heterocyclic group attachedto an aryl group attached to an alkyl-O— group. The heterocyclic, aryland alkyl groups can be optionally substituted as described above.

The term “electrolyte,” as used herein, refers to salts generally andspecifically to ions. An electrolyte refers to an ion that iselectrically-charged, either negative or positive. Common electrolytesinclude chloride (Cl⁻), bromide (Br⁻), bicarbonate (HCO₃ ⁻), sulfate(SO₄ ²⁻), sodium (Na⁺), potassium (K⁺), calcium (Ca²⁺), and magnesium(Mg²⁺).

ABBREVIATIONS

Abbreviations used in the examples are: DCC for1,3-dicyclohexylcarbodiimide; DMAP for 4-dimethylamino-pyridine; LC/MSfor liquid chromatography-mass spectrometer; m-CPBA formeta-chloroperoxybenzoic acid; and PhMe/EtOAC for toluene/ethyl acetate.

Steroidal Quinols

In one embodiment, a quinol of Formula I is provided as follows

or a pharmaceutically acceptable salt or prodrug thereof; wherein thequinol of Formula I is derived from the following estrogen analogue(ZYC1)

ZYC1 is an analogue of estrogen and has been demonstrated to haveestrogen-like activity. The physicochemical properties of ZYC1 inhibitfacile transcorneal penetration upon topical administration (i.e.,eye-drops). In accordance with the present invention, ZYC1 is oxidizedto produce an steroidal quinol,10,17-dihydroxyestra-1,4,9(11)-triene-3-one (“ZYC1-quinol”). TheZYC1-quinol, as discussed in more detail below, has demonstratedimproved physicochemical properties, including decreased lipophilicity,to facilitate transcorneal penetration.

In another embodiment, a quinol of Formula II is provided as follows:

or a pharmaceutically acceptable salt or prodrug thereof, wherein thequinol of Formula II is derived from3-hydroxyestra-1,3,5(10-triene-17-one (estrone).

In yet another embodiment, a quinol of Formula III is provided asfollows:

or a pharmaceutically acceptable or prodrug thereof, wherein the quinolof Formula III is derived from 3,17-dihydroxyestra-1,3,5(10)-triene(estradiol).

The compounds of Formulas I–III can also be functionalized at the 3- or17-keto group as an oxime or alkoxime. Such compounds are useful aspreliminary compounds to the quinol, for use as pro-prodrug compounds.These compounds would be useful for a variety of therapeutic purposesincluding, for example, use as a β-blocker.

The compounds and processes of the invention will be better understoodin connection with the Examples, which are intended as an illustrationof and not a limitation upon the scope of the invention.

EXAMPLE 1 Physicochemical Properties of ZYC1

Human retinal ganglial cells (RGC) were incubated with glutamate (5 mM),the estrogen analogue 3,17-dihydroxyestra-1,3,5(10),9(11)-tetraene(ZYC1) or combination of glutamate and various concentrations of ZYC1.As illustrated in FIG. 1, glutamate killed about 70% of RGC while thecompound of Formula ZYC1 alone had no effect on RGC viability. In thepresence of all three concentrations of ZYC1, glutamate killedsignificantly fewer cells.

RGC were treated with glutamate (5 mM) in the presence or absence ofZYC1. As illustrated in FIG. 2, this estrogen analogue, ZYC1, reducedthe number of RGC killed by glutamate. Where ZYC1 was incubated in thepresence of various concentrations of estrogen receptor antagonist,IC1182,780 (ICI) (which at the lowest concentration used, was more than100-times its IC50), little antagonism of ZYC1 protection of RGC wasseen. This data suggests that ZYC1 protects RGC through a non-estrogenreceptor mediated mechanism. However, the physicochemical properties ofZYC1 permit negligible transcorneal penetration upon topicaladministration.

EXAMPLE 2 Improved Physicochemical Properties of Steroidal Quinols

To test the hypothesis that directed modification of an estrogenimproves physicochemical properties of transcorneal penetration, estronewas used as a lead compound. The following Table I indicates a verysignificant drop in lipophilicity of Formula I, Formula II, and FormulaIII, compared to the parent phenolic A-ring steroids, ZYC1, estrone, andestradiol. The log of the n-octanol/water partitioning coefficient (logP or log D_(7.4)) is the measure of attraction to lipid phase versus anaqueous phase. Log P is a crucial factor governing passive membranepartitioning, influencing permeability opposite to its effect onsolubility (i.e., increasing log P enhances permeability while reducingwater solubility). Thus, the results of Table I demonstrate that thelipophilic-hydrophilic balance of Formula I, Formula II, and Formula IIIare closer to the ideal value for facile transcorneal penetration andfavorable bioavailability than the parent phenolic A-ring steroids,ZYC1, ZYC3, estrone, and estradiol. It has been demonstrated that theideal log P value for the brain is approximately 2. Though an ideal logP value for the cornea has not yet been determined, a log P value of twoshould be a reasonable value for the cornea.

TABLE I COMPOUND Log P P 3-hydroxyestra-1,3,5(10)-triene-17-one(estrone) 4.54 64,670 3,17β-dihydroxyestra-1,3,5(10)-triene (estradiol)4.01 10,230 3,17-dihydroxyestra-1,3,5(10),9(11)-tetraene (ZYC1) 3.573,715 2-(1-adamantyl)-3-hydroxyestra-1,3,5(10)-trien-17-one [2-(1- 6.836.76 · 10⁶ adamantyl)estrone] (ZYC3)10β-hydroxyestra-1,4-diene-3,17-dione (“estrone quinol”) 2.20 15810β,17β-dihydroxyestra-1,4-diene-3-one (“estradiol quinol”) 1.67 4710β,17-dihydroxyestra-1,4,9(11)-triene-3-one (“Formula * quinol”) 1.3020 2-(1-adamantyl)-10β-hydroxyestra-1,4-diene-3,17-dione [“2-(1- 4.3019,953 adamantyl)estrone quinol”] (The log P values pertain to n-octanol/water partitioning were predictedby the method incorporated into CAChe WorkSystem Pro 5.0 (FujitsuAmerica, Inc., Beaverton, Oreg.).

EXAMPLE 3 General Methods for Preparing a Steroidal Quinol

By way of example, Formula II (estrone quinol;10β-hydroxyestra-1,4-diene-3,17-dione) was prepared by the followingScheme I:

As understood by the skilled artisan, steroidal quinols according to thepresent invention may be synthesized using a “one-pot” phenol to quinoltransformation. The synthesis method utilizes m-CPBA as an oxidant,dibenzoyl peroxide [(PheCO)₂O₂] as a radical initiator and visible-lightirradiation that, in refluxing aprotic solvent, produces excellentyields of the quinols of the present invention.

By way of example, Solaja et al., Tetrahedron Letters, 37:21, 3765–3768(1996) discloses a “one-pot” method for synthesizing estrone-quinol.Oxidation of estrone to synthesize 10β-hydroxyestra-1,4-diene-3,17-dioneis performed by heating a stirred solution of estrone (10.00 g, 37.0mmol), m-CPBA (22.53 g, 111.0 mmol; 85% Jansen Chimica), and (PheCO)₂O₂(900 mg, 3.70 mmol) in 2 L mixture of CCl₄/Me₂CO (4/1) to reflux for 3hours while irradiated with a 60 Watt tungsten lamp. Upon evaporation ofthe solvent, extraction is performed with CHCl₃ (3×200 mL), washing withNaHCO₃ (2×100 mL) and H₂O (100 mL), and drying over anhydrous Na₂SO₄.The residue is then chromatographed on SiO₂ column. Elution may beperformed with PhMe/EtOAc (1/1 and 7/3, respectively) andcrystallization from benzene produces 5.19 g (49%) of estrone quinol ascolorless needles.

Data regarding the resulting estrone quinols, as observed by Solaja etal. are as follows: mp=219–221° C. (benzene); ¹H-NMR (250 MHz, DMSO-d₆):7.13 (d, j=10.4 Hz, H—C(1)), 6.07 (dd, J=10.4, 2.4 Hz, H—C(2)), 5.92(irreg. T, J_(4,2)=2.4, J_(4,6β)=1.2 Hz, H—C(4)), 5,67 (s, H-o,exchangeable with D₂O), 2.67 (tdd, J=15.2, 6.4, 1.2 Hz, H_(β)—C(6)),1.97–1.83 (m, H_(β)—C(8) and H_(β)—C(11), 1.30–1.18 (m, H_(α)—C(11)),0.97 (s, H₃C—C(13)); ¹³C NMR (62.9 MHz, DMSO-d₆): 220.33 (C(17)), 185,53(C(3)), 165.09 (C(5)), 150.25 (C(1)), 128.30 (C(2)), 123.09 (C(4)),70.10 (C(10)), 51.18 (C(0), 50.10 (C(14)), 47.75 (C(13)), 35.62 (C(16)),34.58 (C(8)), 32.19 (C(7)), 31.80 (C(6)), 31.03 (C(11)), 22.00 (C(12)),21.90 (C(15)), 13.73 (C(18)); MS (EI, m/z): 286(M⁺, 84), 268(M⁺-H₂O,39), 150(68), 145(100), 124(75), 107(50), 91(50), 79(54), and 55(60).

Alternatively, estrome quinols of the present invention can be preparedusing 2-(1-adamantyl)-3-hydroxyestra-1,3,5(10)-trien-17-one[2-(1-adamantyl)estrone], which can be made using methods previouslydescribed by Lunn, W. H. and E. Farkas, “The adamantly carbonium ion asa dehydrogenating agent, its reactions with estrone,” Tetrahedron,24:6773–6776 (1968). Estrone (270 mg, 1 mmol) and 1-adamantanol (170 mg,1 mmol) were added to anhydrous n-pentane (6 mL) and the stirred mixturewas cooled with an ice bath. Boron trifluoride etherate (BF₃EtOEt, 0.4mL) was added over a 10 minutes period. After an additional 15 min, theice bath was removed and stirring was continued for an additional 45 minat room temperature. During the 45 min, the solid present in thereaction mixture was dissolved and yellow oil formed. Crushed ice wasthen added while shaking and swirling the reaction flask and pink solidwas formed. The filtered crude pink product was washed with water untilthe filtrate had a neutral pH and the solid was dried in a vacuum ovenat 50° C. The pink crude powder (0.4 g) was purified by flashchromatography (silica gel, eluted with 20% ethyl acetate in hexanes toyield the pure product; 0.31 g, 76.7%). The product was recrystallizedfrom a mixture of chloroform and isopropyl alcohol and had: mp 322–324°C., lit mp 295–296° C.; ¹H NMR (CDCl₃, 300 MHz) δ 0.91 (s, 3H, C₁₈—CH₃),2.8 (m, 2H, C₆—CH₂), 4.71 (s, 1H, C₃—OH), 6.42 (s, 1H, Aromatic H), 7.15(s, 1H, Aromatic H).

2-(1-Adamantyl)estrone (also referred to herein as ZYC3) was oxidizedwith lead-acetate to the corresponding quinol acetate using thefollowing procedures. To a solution of 3 g 2-(1-adamantyl)estrone in 50ml of glacial acetic acid 11 g of lead(IV)-acetate was added. Thesolution was stirred at room temperature for 1 day. Then, the solutionwas concentrated in vacuo to an oil that was treated with 50 ml of waterand 50 ml of chloroform. The organic layer was separated and washed with10% NaHCO₃ and water. After drying over Na₂SO₄ the chloroform wasremoved and the residue was purified by column chromatography on silicagel using hexane ethyl acetate 4:1 (v/v) eluent. The pure quinol acetate(as illustrated in FIG. 3), which is also a potential prodrug, hasR_(f)=0.5 on silicagel TLC with the same eluent. Typical resonances(ppm) in ¹H-NMR (CDCl₃) spectrum indicating the conversion to2-substituted estrone quinol acetate were observed: 6.4 (s, 1H, H-1);6.0 (s, 1H, H-4); 2.0 (s, 3H, 10-acetyl).

The quinol acetate (was then hydrolyzed to the quinol (where R═H;prepared as described above) in methanol using a slight excess of NaOMein methanol (25% w/v) overnight at room temperature. Then the solutionwas concentrated and glacial acetic acid was added to adjust the pHslightly acidic. Upon adding water the quinol precipitated out as a paleyellow solid that was again purified by column chromatography the sameway as its acetate (R_(f)=0.53). Typical resonances (ppm) in ¹H-NMR(CDCl₃) indicating the conversion to 2-substituted estrone quinol wereobserved: 6.6 (s, 1H, H-1); 5.9 (s, 1H, H-4). MS (EI): m/z 420 (M^(+•)).

To prepare (alk)oxime estradiol quinols of the subject invention, suchas those illustrated in FIG. 4, 0.5 g of hydroxylamine hydrochloride isadded to 0.5 g of estradiol quinol or alkoxyamine hydrochloride) in 5 mlof ethanol, 0.5 ml of pyridine was added and the solution was refluxedovernight. After cooling, the ethanol was removed and ice-cold water wasadded. The mixture was stirred until the oxime crystallized.

EXAMPLE 4 Physicochemical Properties of ZYC3

RGC were incubated with glutamate (5 mM), with2-(1-adamantyl)-3-hydroxyestra-1,3,5(10)-trien-17-one (ZYC3), or with acombination of glutamate and various concentrations of ZYC3. Asillustrated in FIG. 5, glutamate killed about 70% of RGC while thecompound of ZYC3 alone has no affect on RGC viability. In the presenceof three different concentrations of ZYC3, glutamate killedsignificantly fewer cells (No statistically significant difference fromRGC survival without exposure to glutamate).

EXAMPLE 5 Prodrug Activity

By way of example, conversion of Formula II by NAD(P)H as an endogenousreducing agent was tested. Estrone quinol (0.1 mM) and 1.0 mM of NADPHor NADH in 0.1M sodium phosphate buffer (1 ml final volume, pH 7.5) wasincubated at 37° C. At incremental time points, 100 μl aliquots wereremoved into ice-cold centrifuge tubes, and 100 μl of glacial aceticacid was added. After immediate extraction with ethyl acetate, theorganic layer was evaporated under nitrogen stream. Reconstitution ofthe samples with the liquid chromatography mobile phase was followed byLC/MS analyses, the results of which are illustrated in FIGS. 6–8. Forthe control experiment, no reducing agent was used.

Liquid chromatography separation was done using a Supelco (Bellfonte,Pa.) 5 cm×2.1 mm i.d. Discovery HS C-18 reversed-phase column with 0.25ml/min water:methanol:2-propanol:acetic acid:dichloromethane(53:35:5:5:2, v/v) as a mobile phase. The sample residues were dissolvedin 40 μl of mobile phase, respectively, and 5 μl of the solution wasinjected for analysis. Mass spectra were recorded on a quadrupleion-trap instrument (LCQ®, ThermoFinnigan, San Jose, Calif.) usingpositive-ion atmospheric-pressure chemical ionization (APCI) as themethod of ionization. MS/MS and MS³ product-ion scans were obtainedafter collision-induced dissociation (CID) with helium as the targetgas. Comparison with authentic reference compound (retention time,t_(R), and mass spectra) was used for unambiguous identification ofestrone. As an internal standard,1,3,5(10)-estratrien-17α-ethynyl-17β-ol was added before each sampleextraction. Estrone and estrone quinol levels were determined byLC/APCI-MS/MS and calibration with solutions of known concentrations ofestrone (0.02 μM to 11 μM) and estrone quinol (0.2 μM to 125 μM)extracted for analyses. The chromatographic peak areas for estrone andestrone quinol were obtained from m/z 271→253 and m/z 287→269 MS/MStransitions, respectively. Formation of estrone was clearly detectableeven after a short period of time, when the incubation was carried outin the presence of NADH and, especially, NADPH.

The rate of conversion at 37° C. and with a 10-fold access of theubiquitous reducing agent NADPH is 6.0×10⁻⁷±4×10⁻⁸ M·min⁻¹, whichindicates a rapid process required for the proposed action of a quinolas a prodrug. Enzymes may also catalyze reductions in the eye. See SichiH and D. W. Nebert, “In: Extrahepatic Metabolism of Drugs and OtherForeign Compounds (Gram T E, Ed.),” S.P. Medical and Scientific Books,New York, pp. 333–363 (1980), and Starka L and J. Obenberger. (In vitroestrone-estradiol-17beta interconversion in cornea, lens, iris andretina of rabbit eye,” Arch Klin Exp Ophthalmol, 196:199–204 (1975).

EXAMPLE 6 General Methods for Preparing Prodrugs

In general, where a steroidal quinol according to the subject inventioncontains a hydroxyl group (i.e., 17-OH group or 10β-OH group), an“ester” moiety can replace the hydroxyl portion to form a non-acidic(neutral) ester compound. The addition of a polar functional group(i.e., tertiary amide or phosphate ester) enhances the phenolic A-ringsteroid-derived quinol's affinity to water and thus facilitates thetransport of the quinol through the lipid-poor soma in the cornea. Thefollowing compounds of Formula III, Formula IIIa and Formula IIIb,illustrate polar functional groups attached at the 17-OH group.

wherein

each R and R′ is independently hydrogen, alkyl, alkenyl, alkynyl,alkoxy, aryl, aralkoxy, aralkyl, aryloxy, hydroxyalkyl, alkoxyalkyl,heteroaralkyl, heterocyclealkyl, heteroaryloxy; and heterocyclearalkoxy;

X is an electrolyte; and

n is an integer from 1 to 20.

By way of example, a prodrug of Formula I (n=1, R═H) may be obtained byconverting Formula III into an ester compound as illustrated in thefollowing Scheme IIa. To a solution of10β,17β-dihydroxyestra-1,4-diene-3one (Formula I, estradiol quinol) inchloroform or ethyl acetate bromoacetic anhydride, DCC, and DMAP areadded. The resulting mixture is stirred at 20–25° C. for 48 hours. Theorganic solution is extracted with water then dried over Na2SO4 andevaporated. The residue is purified by chromatography (silica gel:Aldrich, Merck grade 60, 230–400 mesh, 32×2 cm; elution with hexanecontaining gradually increasing concentrations of ethyl acetate from 0to 6%). The purified residue in hexane is then placed in a closed systemunder argon, and trimethylamine (gas) was added at 20–25° C. then theprecipitate was filtered, and rinsed with hexane. The resultant prodrugof estradiol quinol(10β,17β-dihydroxyestra-1,4-diene-3-one-17-acetyl-trimethylammoniumbromide) should have adequate solubility and sufficient stability toallow for formulation and storage. Further, the exemplary prodrug ofestrone quinol is easily converted through an enzymatic or chemicalprocess to the active compound, estrone, within the body, preferably theeye. In the following Scheme IIa, a prodrug of Formula I can be obtained

wherein R, R′, H, and n are as defined above.

Phosphate esters can also be attached as a polar functional group toenhance water affinity of steroidal quinols. For example, phosphateester prodrugs of estrogens according to the present invention can beprepared by an ester linkage to one of the hydroxyl groups of the headgroup of an steroidal quinol.

By way of example, the prodrug of estradiol, in accordance with thepresent invention, may be prepared using general methods as depicted inthe following Schemes IIIa and IIIb.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

1. A method for treating a patient diagnosed with at least oneophthalmic disorder, wherein said method comprises administering to thepatient an effective amount of a steroidal quinol that is converted to abiologically active phenolic A-ring steroid compound in vivo, whereinthe steroidal quinol has the general structure:


2. The method, according to claim 1, further comprising administeringthe quinol by a route selected from the group consisting of oral,topical, buccal, intramuscular, transdermal, intravenous, andsubcutaneous.
 3. The method, according to claim 1, wherein theophthalmic disorders are selected from the group consisting ofconjunctivitis, diabetic retinopathy, dry eye, macular degeneration,glaucoma, and cataracts.
 4. The method, according to claim 2, whereinthe steroidal quinol is administered topically in the form of eye dropsor membranous ocular eye patch.
 5. A method for treating a patientdiagnosed with at least one ophthalmic disorder, wherein said methodcomprises administering to the patient an effective amount of asteroidal quinol that is converted to a biologically active phenolicA-ring steroid compound in vivo, wherein the quinol has the generalstructure:


6. The method, according to claim 5, further comprising administeringthe quinol by a route selected from the group consisting of oral,topical, buccal, intramuscular, transdermal, intravenous, andsubcutaneous.
 7. The method, according to claim 5, wherein theophthalmic disorders are selected from the group consisting ofconjunctivitis, diabetic retinopathy, dry eye, macular degeneration,glaucoma, and cataracts.
 8. The method, according to claim 6, whereinthe steroidal quinol is administered topically in the form of eye dropsor membranous ocular eye patch.
 9. A method for treating a patientdiagnosed with at least one ophthalmic disorder, wherein said methodcomprises administering to the patient an effective amount of asteroidal quinol that is converted to a biologically active phenolicA-ring steroid compound in vivo, wherein the steroidal quinol is2-(1-adamantyl-10β-hydroxyestra-1,4-diene-3,17-dione.
 10. A steroidalquinol that is 2-(1-adamantyl-10β-hydroxyestra-1,4-diene-3,17-dione. 11.A pharmaceutical composition comprising2-(1-adamantyl-10β-hydroxyestra-1,4-diene-3,17-dione.